Selective Plane Illumination Microscopy (SPIM) and its variants Ultramicroscopy, Objective-Coupled Planar Illumination Microscopy (OCPI), and Oblique Plane Microscopy (OPM) are approaches that can use light sheet imaging to image fluorescence contrast. Such approaches can provide diffraction or near diffraction limited resolution across 3D volumes. However, it is believed that these approaches require rigid alignment between the light sheet used for illumination and the camera used for detection. This makes it necessary to either translate the entire imaging system or the sample relative to the light sheet for 3D imaging.
Another approach, Optical Projection Tomography (OPT) can provide volumetric imaging based upon projection imaging using light. In this approach, light is shone directly through the sample and detected collinearly, in an analogous imaging technique to x-ray Computed Tomography (CT). The sample, or the complete imaging system must be physically rotated using a precision rotation stage to acquire projection images. A modified back-projection algorithm is then used to generate 3D images of absorbing or fluorescent contrast.
The present inventors have recognized that one problem with OPT and SPIM-type imaging approaches is that such techniques are limited to minimally or non-scattering samples or to larger ex-vivo, chemically cleared tissue samples. Additionally, the need for precise sample translation and/or rotation for OPT and SPIM significantly limits the rate at which these techniques can scan 3D volumes and the types of samples that can be imaged.
Laminar Optical Tomography (LOT) is an optical imaging approach that takes advantage of photon scattering in tissues by detecting backscattered light which has travelled to different depths within a sample. LOT can provide non-contact, scanning spot imaging to depths of up to 2 mm in living tissues and has recently been extended to allow simultaneous multispectral imaging of both absorption and fluorescence contrast. An example of LOT is described in Hillman et al. U.S. patent application Ser. No. 12/655,325, entitled OPTICAL IMAGING OR SPECTROSCOPY SYSTEMS AND METHODS,” filed on Dec. 29, 2009, which published on Jul. 1, 2010 as U.S. Patent Application Publication No. US-2010-0168586-A1, each of which is incorporated by reference herein in its entirety. However, owing to its illumination and detection geometry, such LOT techniques are primarily useful to image scattering samples, as it is particularly configured to detect photons which have backscattered from the tissue.